Study On The Effect Of Estradiol Supplementation On Endometrium Receptivity During The Luteal Phase In Controlled Ovarian Stimulation Cycles

1.BackgroudThe disease incidence of infertility is about 10～15%in women of reproductive age,and it does harm to the harmony of family and society.There are four factors that are essential to the success getting pregnancy,they are sperm and ovum of high-quality, unobstructed fallopian tubes,receptive endometrium.Endometrium is the "soil" for embryo implantation,so it is very important to successful implantation.The human endometrium becomes receptive to the embryo only for a limited period during the luteal phase of the menstrual cycle,the period during which endometrium is receptive to implantation,termed the implantation window,begins approximately 5-10 days after ovulation and is believed to encompass cycle days 19-24.Numbers of morphological and molecular markers specific to the implantation window has been identified,including endometrial morphological features,pinopodes and a large number of identified molecular mediators under the influence of ovarian hormones.The recent discovery of molecules crucial for successful embryo implantation has offered researchers precious insight into this field,for example, leukaemia inhibitory factor(LIF),integrins,Interleukin(IL)-1,epidermal growth factor(EGF),CSF-1,estradiol receptor,progesterone receptor,et al.Nowadays,with the development of in vitro fertilization-embryo transfer (IVF-ET)used in infertility,people can achieve high ovulation rates by choosing different super-ovulation protocols.However,implantation rates per embryo transfer is still very low.There are many factors affecting successful implantation,but the endometrial receptivity and embryo quality are two of the crucial factors for successful implantation.A large number of identified molecular mediators,under the influence of ovarian hormones,have been postulated to be involved in this early stage of endometrial receptivity.In IVF-ET cycles,patients undergoing COS became a common practice.Use of GnRH agonists in ovarian stimulation protocols to suppress premature LH surges increased the number of mature oocytes and embryos available for transfer and subsequently improved pregnancy rates.GnRH agonist use,however, has been associated with several undesired effects.Premature luteolysis and inadequate endometrial priming because of oversuppression of pituitary function are some of the unwanted consequences.It is generally accepted that those reduce uterine receptivity and,thus,the chances of conception.The necessity of luteal-phase support after GnRH agonists has been generally accepted.Luteal-phase support was routinely either in the form of progesterone, despite limited information about the quality of the luteal phase.Thus far,studies comparing different luteal support protocols in women undergoing ovarian stimulation used implantation and pregnancy rates as endpoints.Recently,there are data to support an improvement in implantation and pregnancy rates in the groups that had received a combination of estrogens and progesterone for luteal-phase support, but this is not a common practice by all.Whether this contributes to an improvement in the endometrial receptivity and subsequent implantation and pregnancy rates is unclear.Untill now,there are no study on molecular factors,endometrial morphology and clinical data to find whether estrogen supplementation for luteal-phase support will improve the endometrial receptivity.2.ObjectiveIn the present study,we evaluate the impact of different types of luteal-phase support after ovarian stimulation with a GnRH agonists protocol on the endometrial receptivity.In view of this,the objectives of the current research were to test the expression regulation of LIF and LIFmRNA,pinopod in peri-implantation mouse endometrium.Then study the effects on their expression as well as human implantation rate by setting up animal model based on different treatment protocols. The patients underwent long protocols for controlled ovarian stimulation for IVF treatment with GnRH agonist suppression were studied retrospectively.To study the effect of the serum estradiol level down-range on the day of embryo transfer in In-Vitro fertilization out- comes and the necessity and the effect of individual estradiol supplementation during the luteal phase in In-vitro fertilization outcome.In a serial study,we try to evaluate the relationship of LIF,pinopode and implantation,the molecular mechanism of negative effects based on different treatment protocols.These studies provide a new theory basis for optimizing the treatment protocols to improve the In-vitro fertilization outcomes.These studies are divided into three parts as listed below:3.Material and methods3.1 Material and methods of animal experiments3.1.1 GroupingFemale mice were randomly divided into four groups:group A,group B and group C were controlled ovarian stimulation(COS) groups;Group D was nature cycle group. Female mice in COS groups were rendered pregnant by mating with male mice after HCG.The next morning,the vaginal plugs were examined.Pregnancy day 1 was determined on the vaginal plug day.3.1.2 Medication programFemale mice in COS groups were divided into three groups according the different types of luteal-phase support.Group A:without hormonal support of the luteal phase in female mice undergoing COS,Group B:with progesterone for luteal-phase-support;Group C:with progesterone and estradiol supplementation for luteal-phase-support.3.1.3 Tissue samples collection Tissue samples were collected from pregnant mice on day3,day4,day5 of vaginal plugs day.3.1.4 scanning electronic microscopyPinopodes were investigated by scanning electronic microscopy(SEM) in the uterine endometrial of pregnant mice.3.1.5 LIF protein was determined by immunohistochemistry in the uterine endometrial of pregnant mice.3.1.6 The LIF mRNA expression was measured by reverse transcriptional-polymerase chain reaction(RT-PCR) in the uterine endometrial of pregnant mice.The levels of LIF protein and LIFmRNA in the endometrium were analyzed by semi-quantitative assay.3.2 Clinieal Material and methods3.2.1 In this study,97 IVF-ET cycles in long protocol were evaluated retrospectively. Serum estradiol was measured on the day of HCG administration and the day of embryo transfer(ET) by chemiluminescence technique.They were divided into six groups according to the down-range of serum estradiol level at the ET day,group one (＜20%),group two(20%-29%),group three(30%-39%),group four(40%-49%), group five(50%-59%),group six(≥60%).To study the effect of the serum estradiol level down-range on the day of embryo transfer in In-Vitro fertilization outcomes.3.2.2 To study the necessity of estradiol supplementation,216 long protocol COS and IVF-ET cycles were evaluated retrospectively.They were divided into two groups according the methods of the luteal phase surport.Group A₁ 97 cycles received only progesterone as the luteal phase surport.Group B₁ 119 cycles received the same dosage of progesterone,combined with oral E₂ 3mg/d to the patients who the down-range of serum E₂ level at the ET day were≥30%.3.2.3 To study the effect of individual estradiol supplementation,104 long protocol COS and IVF-ET cycles were evaluated retrospectively.They were divided into four groups according the methods of the luteal phase surport.Group A₂ 12 cycles received only progesterone as the luteal phase surport.Group B₂ 18 cycles received the same dosage of progesterone,combined with oral E₂ 3mg/d to the patients who the down-range of serum E₂ level at the ET day were 30%-39%.Group C 16 cycles received the same dosage of progesterone combined with oral E₂ 4mg/d to the patients who the down-range of serum E₂ level at the ET day were 40%-49%.Group D 58 cycles received the same dosage of progesterone combined with oral E₂ 4mg/d to the patients who the down-range of serum E₂ level at the ET day was≥50%.3.2.4 To compare the embryo implantation rate and clinical pregnancy rate in estradiol supplementation luteal-phase support protocol with classical luteal-phase support protocol.3.2.5 To compare the embryo implantation rate and clinical pregnancy rate in different estradiol supplementation protocols.3.3 Statistical analysisStatistical analyses were performed by running the SPSS11.0 software packages. Student's two-tailed t test was then used to compare the two study groups on normally distributed variables,P values less than 0.05 were used to define statistical significance.4.Results4.1 Results of animal experiments4.1.1 Results of pinopodes in endometrialIn group B,C,D,there were small developing pinopodes in pregnant mouse endometrial surface on day 3;there were large fully developed pinopodes in endometrial surface,which was smooth with well defined borders resembling a mushroom on day 4 of these group;the regressing pinopodes were observed on day 5 of these group.Pinopodes in group D and group C were better than in group B.In group A,there were small developing pinopodes in pregnant mouse endometrial surface on day 3;the regressing pinopodes were seen on day 4 of group A.4.1.2 Qualitation assay the expression of LIF proteinIn the pregnant mouse of group C and D,weak immunostaining of LIF protein in endometrial epithelial cell and glandular epithelial cell was found on day 3 of gestation,strong immunostaining(++) peaks on day 4,and then immunostaining (+)peaks on day 5;in the pregnant mouse of group B,weak immunostaining of LIF protein in endometrial epithelial cell and glandular epithelial cell was found on day 3 of gestation,immunostaining(+)peaks on day 4,and then weak immunostaining on day 5;in the pregnant mouse of group A,weak immunostaining of LIF protein in endometrial epithelial cell and glandular epithelial cell was found on day 3 of gestation,weaker immunostaining on day 4 and day 5.4.1.3 Semi-quantitative assay of LIF proteinThe results of statistical analyses showed that:in the pregnant mouse of group C and D,the level of LIF protein on day 3-5 was significantly higher than group A(P＜0.05);in group B,the level of LIF protein on day 3-5 was significantly higher than group A(P＜0.05),but significantly lower than group C and D(P＜0.05).The expression of LIF was no different between group C and D.4.1.4 Comparing the expression of LIF mRNA between different days in every groupIn group B,C,D,the expression of LIF mRNA in pregnant mice endometrium were positive;the expression of LIF mRNA in pregnant mouse endometrium of group A were very weak.The results of semi-quantitative assay showed:the LIF protein surges of pregnant mouse in group B,C,D wereon day 4;but in pregnant mouse endometrium of group A,the expression of LIF protein was stronger immunostaining on day 3,and weaker immunostaining on day 4 and day 5.4.1.5 Comparing the expression of LIF mRNA between different groupsThe results of semi-quantitative assay of LIF mRNA in pregnant mouse endometrium showed:compared with group A,the level of LIF mRNA was significantly higher in the pregnant mouse of group B,C and D(P＜0.05);compared with group B,the level of LIF mRNA was significantly higher in the pregnant mouse of group C and D(P＜0.05);there were no significantly different between group C and D.4.1.6 The peak expression of LIF mRNAThe results showed that:the expression ofpinopodes,LIF protein and LIF mRNA in group D and group C were better than in group B on day 4;and compared with COS group the expression of those were the weakest in group A on day 3.The results of semi-quantitative assay and statistical analysis showed:compared with group A,the levels of LIF mRNA and LIF protein were significantly higher in the pregnant mouse of group C and group D(P＜0.05);in group B,the levels of LIF mRNA and LIF protein were higher than in group A(P＜0.05),but were significantly lower than in group C and group D(P＜0.05);there were no significantly different between group C and D.4.2 Clinical results4.2.1 The estradiol concentration had the different down level on the day of embryo transfer.4.2.2 The higher of the down-range of serum E₂ level,the lower of the embryo implantation and pregnancy rate.When the down-range of the estradiol on ET day was more than 30%,the embryo implantation rate and the clinical pregnancy rate were significantly lower(P＜0.05).4.2.3 The down-range of the estradiol on ET day was more than 30%In 74.23%of all cycles.4.2.4 The older patients had higher down-range of serum E₂ level,but there were no significantly different.4.2.5 Comparing the parameters between different groupsThere were no differences on average age,endometrium thickness,fertiliz -ation rates,embryo cleavage rates,and quantity or quality of embryos transferred between Group A₁ and Group B₁;there were also no differences among Group A₂,Group B₂,Group C and Group D.4.2.6 Comparison between group A₁ and group B₁In Group A₁ when the down-range of E₂ on ET day was more than 30%,the embryo implantation rate and the clinical pregnancy rate were significantly lower (P＜0.05).In Group B₁ when the down-range of E₂ was more than 40%,the embryo implantation rate and the clinical pregnancy rate were also significantly lower (p＜0.05).4.2.7 Comparison between different doses of estradiol supplementation Different doses of estradiol supplementation were given according to different down-range of the serum E₂ level,when the down-range of the estradiol on ET day was more than 40%,there were no significant differences on the embryo implantation rate and the clinical pregnancy rate(P＞0.05);when the down-range of the estradiol on ET day was more than 50%,the embryo implantation rate and the clinical pregnancy rate were significantly lower(P＜0.05).5.Conclusions5.1 The expression of pinopodes,LIF protein and LIF mRNA are weaker in COS group,and the implantation window opens earlier.5.2 Luteal-phase support with progesterone could improve the endometrial receptivity greatly in COS group.5.3 Compared with luteal-phase support with progesterone only,estradiol supplementation could improve the expression of pinopodes,LIF protein and LIF mRNA in mouse endometrium in COS group,and then improve the endometrial receptivity.5.4 In long protocol COS and IVF-ET cycles,the rapidly decline of the serum estradiol has the important effect to the IVF-ET outcome.When the down-range of the serum estradiol is more than 30%from HCG day to ET day,the embryo implantation rate and clinical pregnancy rate are lower.5.5 There are 74.23%of all cycles with the down-range of the serum E₂ level more than 30%.5.6 The older patients have higher down-range of serum E₂ level.5.7 In long protocol COS and IVF-ET cycles,the estradiol supplementation was necessary during the luteal phase surport to the patients whose down-range of E₂ level on ET day is more than 30%.5.8 The dose of the estradiol supplementation should be individual based on the down-range of the serum E₂ level.